The major long-term objective of our research is to understand the detailed molecular mechanisms by which proteins recognize and interact with specific DNA sequences. This problem is being approached through a detailed structural and functional study of type II restriction and modification systems. The EcoRI system is the best characterized. The gene and amino acid sequences of both enzymes have been determined, and X-ray crystallographic analysis is beginning to yield detailed information about the structure of the endonuclease-DNA complex. Complementary contacts between the enzymes and their substrate sequence will be explored. Mutants of the EcoRI endonuclease will be isolated and mapped. Altered enzymes will be purified and analyzed for functional parameters such as kinetics of DNA hydrolysis, stability of dimer structure, equilibrium binding constants and contacts with the DNA substrate. Other type II systems will be examined in order to determine the generality of information derived from the EcoRI system. In particular, an isoschizomer of EcoRI has been identified in Rhodopseudomonas sphaeroides. Although R. sphaeroides and E. coli are rather distantly related, preliminary experiments indicate that the endonucleases have a common origin. The RsrI restriction and modification system will be cloned into E. coli. DNA sequence analysis will be used to deduce the amino acid sequences of the enzymes. The enzymes will be purified and functional parameters will be compared to the EcoRI counterparts. Comparative data from both of these systems will be analyzed in relation to the three-dimensional structure of the wild type enzyme-DNA complex derived by X-ray crystallographic analyses. Recognition of defined sequences in DNA is an essential process in DNA replication, recombination and the expression of the genome during cellular metabolism and development. Elucidation of mechanisms involved in sequence-specific interactions utilizing relatively simple systems should illuminate these complex processes. The mode of gene regulation utilized by type II restriction-modification systems has not been elucidated for any system. In the case of the EcoRI system, it has been shown that the methylase is required to activate ezpression of the endonuclease. The mechanism of this activation has not been identified. Beta-galactosidase fusion plasmids will be used to monitor methylase activation. In vivo transcription will be analyzed by Northern blot analysis and S1 mapping.